Recombinant herpesvirus of turkeys and use thereof

ABSTRACT

The present invention provides a recombinant herpesvirus of turkeys modified by the presence of cDNA encoding the F protein of Newcastle disease virus under the control of a promoter. The poultry vaccine consisting of the recombinant herpesvirus of turkeys of the present invention can induce in chickens protective immunity against Newcastle disease virus.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention provides a recombinant herpesvirus ofturkeys (rHVT) comprising cDNA of the F protein (F gene) of Newcastledisease virus (NDV) under the control of a modified chicken beta-actinpromoter.

[0003] 2. Description of the Related Art

[0004] Newcastle disease is one of the most fearful contagious diseasesin the poultry industry. Newcastle disease presents itself in many formsranging from high mortality to an asymptomatic form. Strains areclassified as (1) velogenic (high-virulence), (2) mesogenic(moderate-virulence), (3) lentogenic (low-virulence), and (4)asymptomatic (Alexander, D. J. In Diseases of Poultry 1997). Chickensinfected with velognenic forms of NDV become gloomy and lethargic in afew days and the mortality rate is from dozens to more than fiftypercent. Surviving birds often develop neurological symptoms such aswryneck or gyrospasm. One of the reasons the disease is so fearsome isthat chickens are susceptible to NDV regardless of age and thoseinfected with velogenic NDV show fulminant symptoms at all ages. SinceNDV is highly transmissible, every chicken must be disposed of at theoutbreak of the disease. The hennery should thoroughly be disinfected toprevent further infection. Strains in the mesogenic pathotype(moderate-virulence) are characterized by death in young chickens.Strains in the lentogenic pathotype (low-virulence) are characterized bymild respiratory infections and many of these strains are used toprepare vaccines for use in young chickens. Asymptomatic enteric strainsare usually isolated from the gut of chickens showing no disease(Alexander, 1997). Newcastle disease affects both chickens and turkeys,however the clinical signs in turkeys are less severe than in chickens(Alexander, 1997).

[0005] At present, live and inactivated vaccines are available for theprevention of Newcastle disease. These vaccines are effective but notfree from defect. The inactivated vaccine must be inoculated intobreeder hens in lay, repetitively. The live vaccine is mainly for youngchickens. However, long lasting immunity is not guaranteed for youngchickens, which have high maternal antibody levels. Repeatedadministration of live vaccines is sometimes detrimental to the healthygrowth of young chickens due to vaccine reactions causing mildrespiratory disease. Thus, a new type of vaccine, which is efficacious,free from adverse effects, and does not require repeated administration,is desirable for the poultry industry.

[0006] To meet the industry's wishes, Sakaguchi et al. (J. Virol.74:3217-3226,2000; Vaccine 16:472-479, 1998) developed a recombinantMarek's disease virus serotype 1, which had the NDV F gene in the US10region of the virus genome. The obtained recombinant virus inducedlasting protective immunity against Newcastle disease in SPF chickens aswell as in maternal antibody positive commercial birds.

[0007] Marek's disease is another serious problem for the poultryindustry. Against this disease, herpesvirus of turkeys (HVT) (Witter R.L. et al. Am. J. Vet. Res. 1970, 31, 525-538) has been most widely usedas a safe vaccine.

[0008] Until now, there have been several reports about the HVT-basedNewcastle disease-Marek's disease bivalent vaccine. For instance, Morganet al. (Avian Dis. 37:1032-1040, 1993; Vaccine 11:349-358, 1993; AvianDis. 36:858-870, 1992) constructed recombinant HVTs having the NDV Fgene and examined the efficacy of these recombinants as a Newcastledisease vaccine. Macmillan et al. (Vaccine 14:469-477, 1996) constructedrecombinant HVTs expressing HN and F proteins of NDV and tested theserecombinants for efficacy. In both cases, vaccinated SPF chickens wereprotected against NDV, but not satisfactorily commercial chickens havinghigh maternal antibody levels

[0009] Saitoh et al. inserted cDNA encoding F and HN proteins of NDVinto the HVT genome (WO 99/18215). The inserted genes were under thecontrol of the CMV or RSV promoter. The foreign gene insertion site wasa newly identified intergenic region between UL44 and 45 or between UL45and 46. These recombinants conferred good protection against NDVchallenge in SPF chickens as well as in chickens with NDV maternalantibodies. However, these recombinants expressed two inserted genes ofHN and F. Since HN protein induces heamagulutination inhibition (HI)antibodies to immunized chickens, it is difficult to distinguish vaccineimmunized chickens and NDV infected chickens. Therefore, a recombinantvirus expressing F protein gene, which induces an adequate protectiveimmunity against Newcastle disease, is a more desirable vaccine. Thisobjective was not easily attainable because rHVT having only F gene ofNDV didn't induce desirable immunity in chickens as indicated by Morganet al.

SUMMARY OF THE INVENTION

[0010] The present invention provides a recombinant herpesvirus ofturkeys modified by the insertion of cDNA of the F protein of NDV underthe control of a modified chicken beta-action promoter (Pec promoter).The rHVT induces long lasting protective immunity against NDV in SPFchickens as well as in commercial chickens that have high maternalantibody to NDV.

[0011] Specifically, the present invention provides a recombinantherpesvirus of turkeys modified by the insertion of the NDV F gene underthe control of a promoter, of which sequence is described in SEQ NO.1.The present invention further provides a Newcastle Disease-Marek'sDisease bivalent vaccine consisting mainly of the said recombinantherpesvirus of turkeys.

[0012] The present invention is described below in more detail. SEQ IDNO.1 5′-AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATTGGCCCCGCCGGCTGACCGCCCACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCATTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGATGCAGTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGGCGGGGCGGGGCGAGGGGGGCGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCT GAGTGACCGCGTCTAGAGG3′(NDV F gene)

[0013] As long as encoding the NDV F protein, any gene from any NDVstrain is appropriate for the purpose of the present invention. The Fgene of Sato, Miyadera, D26, Atami, or Fuji as well as of Texas GB, B1,or LaSota strain is an example. The F gene of a field isolate or of anyknown DNA sequence is also appropriate. Among these, the gene from D26is a favorable example. Incorporating an additional antigen gene intothe backbone virus is not desirable for the purpose of the presentinvention.

[0014] (Promoter)

[0015] In the present invention, the NDV F gene is controlled by apromoter described in SEQ NO.1 (designated Pec promoter) or thathomologous to it. The Pec promoter (Japanese Unexamined PatentPublication No. 2001-188) is generated by deleting a dispensable regionof the chicken beta-actin promoter. A promoter homologous to Pec means apromoter of which activity is nearly equal to that of Pec and of whichlength is 120 to 850 base pair (bp) or more favorably 150 to 600 bp. Ahomologous promoter can be generated by substitution, deletion, oraddition of nucleotides of to the beta-actin promoter. The promoter usedin the present invention may include a naturally occurring or modifiedenhancer sequence. An example of such a promoter is COA promoterdescribed in SEQ NO.2.

[0016] (Avian Herpesvirus)

[0017] As long as being non-pathogenic to chickens, any herpesvirus ofturkeys can be used in the present invention. For instances, FC126 (ATCCVR-584B), PB-THV1, H-2, YT-7, WTHV-1, or HPRS-26 strain is suitable forthe backbone virus. Among these, FC126 is favorably used in the presentinvention because of its safe use in chickens.

[0018] (Region for Gene Insertion)

[0019] Several non-essential regions of HVT are known, which aredispensable for virus growth and suitable for NDV F gene insertion. Forinstance, UL43 (WO 89/01040), US2 (WO 93/25665) or inter-ORF regionbetween UL44 and UL46 (WO 99/18215) is appropriate for the insertion ofthe F gene. Among these, the inter-ORF region between UL44 and UL46 ismost suitable.

[0020] For the present invention, a non-essential region can newly beidentified by the following general procedure. First, avian herpesvirusDNA fragments of appropriate length are cloned into an E. coli plasmidand physically mapped by restriction enzyme analysis. Then, a genecassette consisting of a promoter and a marker gene is inserted into anappropriate restriction site of the cloned DNA fragment resulting in ahomology plasmid. As described later, if the homologous recombinationwith the obtained homology plasmid resulted in a recombinant virusexpressing the inserted marker gene and if it were stable in vitro andin vivo, the originally selected DNA fragment should be a non-essentialregion suitable for NDV-F cDNA insertion.

[0021] (Construction of rHVT)

[0022] For the present invention, any known method of generating therecombinant avian herpesvirus is applicable. A typical example is asfollows. (1) First, as described above, a recombinant plasmid isconstructed, which includes a non-essential region of the avianherpesvirus. Then, preferably with a promoter at the 5′ terminus and apolyadenlyation signal at the 3′ terminus, NDV-F cDNA is inserted intothe said non-essential region to generate a homology plasmid. (2) Theresultant plasmid is transfected into chicken embryo fibroblast (CEF)cells infected with parent HVT or co-transfected into CEF cells withinfectious HVT genomic DNA. Transfection is performed by any knownmethod (3) The transfected CEF cells are inoculated into culture platesand incubated till the virus plaques become visible. (4) Theidentifiable plaques include recombinant viruses as well as parentwild-type viruses. The recombinant virus is purified from wild typevirus by any known method to screen expression of inserted foreigngenes.

[0023] (Newcastle Disease-Marek's Disease Bivalent Vaccine)

[0024] Since the F protein is a protective antigen of NDV and thebackbone HVT is a live Marek's disease vaccine, rHVT containing the Fgene of the present invention may be used bivalent vaccine againstNewcastle and Marek's diseases or as monovalent vaccine againstNewcastle disease.

[0025] The vaccine consisting mainly of rHVT of the present inventionmay include chicken cells and/or ingredients of culture media. As longas not pharmacologically detrimental, the vaccine may contain anyingredients such as preservatives. In addition, the vaccine of thepresent invention can be used as a mixture with any recombinant ornon-recombinant viruses such as the MDV serotype 1 or serotype 2 vaccinestrains.

[0026] Any known method is applicable to the preparation of therecombinant bivalent vaccine of the present invention. For instance,rHVT is inoculated into permissive culture cells such as CEF cells andgrown to an appropriate titer. Then, the cells are scraped off fromculture plates or bottles by scraper or by trypsin treatment andsubjected to centrifugation. Cells separated from the supernatant arethen suspended in the culture medium containing dimethyl sulfoxide andstored in liquid nitrogen. When rHVTs are in the supernatant, they arecollected and lyophilized.

[0027] The bivalent recombinant HVT vaccine is administered to chickensby any known method of inoculating Marek's disease vaccine. Forinstance, the vaccine of the present invention is diluted to give10-10⁵, or more favorably 10²-10⁴ plaque forming units (PFU)/dose, andinoculated into subcutaneously behind the neck of one day of agechickens or into embryonating eggs by syringe or by any apparatus forinjection.

[0028] The present avian bivalent vaccine gives SPF chickens 90% or moreprotection against the NDV challenge and at least 70% or more protectionto the commercial chickens having a significant level of anti-NDVmaternal antibody.

[0029] In the present invention, protection against NDV challenge isdetermined by the ratio of protected birds to total tested birds in thechallenge testing as described in the examples. First, the appropriatedose of the NDV challenge virus is determined by challengingnon-vaccinated birds. 90% or more of these birds (the negative controlgroup) must show clinical signs. Next, the vaccinated birds arechallenged with the same dose of the challenge virus by intra-muscularroute to the femoral region, or by intra-tracheal, intra-ocular, orinfraorbital sinus route. The challenged birds were observed for onsetof Newcastle disease, specificly neurological symptoms.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 Construction of theHomology Vector

[0030] The plasmid construction was essentially performed by thestandard molecular biology techniques (Molecular Cloning: A LaboratoryManual. 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. 1989). DNA restriction fragments were electrophoresed on agarosegels and purified with QIAquick Gel Extraction Kit (QIAGEN, Cat #28704).

[0031] 1-1 Construction of the Plasmid Used for the Measurement of thePromoter Activity

[0032] Plasmid pGIMICSpolyASfi (2773 bp, WO 99/18215) was generated byintroducing a polyadenylation signal and aSfiI site into themulti-cloning site of pUC18. pGIMICSpolyASfi was then digested with NheIand ligated with the 1721 bp fragment which was excised from Pica geneEnhancer2 (5064 bp, TOYO INK MFG. CO., LTD.) by NheI and XbaI digestion.The obtained plasmid was designated pLUC-Pro (4494 bp).

[0033] Next, a DNA fragment (about 1.5 kilobases) including thebeta-actin promoter was prepared by PCR using a chicken cell genomic DNAbank as a template. The primers were PrBac1 (SEQ ID NO. 2,5′-CAGTGTCGCTGCAGCTCAGTGCATGCACGCTCATTGCCC-3′) and PrBac2 (SEQ ID NO.3,5′-GCTCTAGAGTCGACAAGCTTCATGGCTGGCTGCGGAGGAACAGAGAAGGG-3′).

[0034] The obtained fragment was digested with PstI and XbaI and ligatedwith the 4482 bp fragment generated by digesting pLUC-Pro with PstI andXbaI. The resultant plasmid, designated pLUC-bac, was 5986 bp long andincluded the beta-actin promoter region.

[0035] pLUC-bac was digested with BamHI and BglII and the resulting 5958bp fragment was self-ligated to yield pLUC-bac-Sma. Since pLUC-bac-Smahad PstI and SmaI sites upstream of the beta-actin promoter, restrictionof pLUC-bac-Sma with PstI and SmaI followed by self-ligation yielded thefirst plasmid to obtain the shortened 5′-region of the beta-actinpromoter. Similarly, restriction of pLUC-bac with SacI and XbaI followedby self-ligation generated the second plasmid to obtain the shorted3′-region of the beta-actin promoter. The resultant two plasmids werethen linearized and subjected to the ExoIII treatment usingKilo-Sequence Deletion Kit (TAKARA SYUZO CO., LTD.). Sampling wasperformed at one, two, and three minutes to obtain 7 samples from thefirst plasmid and 22 from the second. All 29 fragments included about200 to 1500 bp promoter region. The obtained fragments were blunt-endedand self-ligated by the standard procedure to generate the circularplasmids.

[0036] The measurement of the luciferase activity was performed withPicaGene (TOYO INK MFG. CO., LTD.). Briefly, one μg each of 29 plasmidswas introduced into CEF cells by electroporation using Gene Pulser(Bio-Rad Laboratories). Cells were then cultured for 24 hours, and celllysis buffer (Included in PicaGene kit) was added. The cells were frozenfor one hour at −80° C., and thawed at room temperature to comp ete thecell lysis. After centrifugation, 10 μl of the recovered supernatant wasadded to 100 μl of the PicaGene substrate solution and one minute later,intensity of luminescence (Unit: RLU) was measured with Lumino-Meter(Model 11253, Bio-Rad Laboratories). The Lumino-Meter was used tocalculate the amount of the produced luciferase, an indicator of thepromoter activity (see FIG. 1).

[0037] As shown in FIG. 1, among the samples having a deletion towards5′-terminus, those lacking more than 120 bp upstream of the TATAAA boxshowed a drastic decrease in promoter activity. On the contrary, therewas no co-relation between the promoter activity and the length of thedeletion towards the 3′-terminus. For instance, a deletion of about 100bp incurred about a one-fifth decrease in the promoter activity whencompared with that of the full length beta-actin promoter. Furtherdeletion (greater than 100 bp) towards the 3′-terminus did not regainthe original activity.

[0038] 1-2 Core Sequence Promoter

[0039] References from the literature (Ref. 12-19) indicate all regionsof the beta-action promoter are not indispensable. Based on thisinformation, 273, 211, 175, and 163 bp fragments were obtained by PCRusing the beta-actin promoter of pLUC-bac as a template. Primers wereSEQ NO. 4 (5′-TATTTTGTGCAGCGAT-3′) and SEQ NO. 5(5′-ACGTCTAGAAGGCAACGCAGCGACT-3′), or SEQ NO. 4 and SEQ NO. 6,(5′-CTGTCTAGATAACGCGGTCAGTCAGA-3′). The obtained fragments coa273,coa211, coa175 and coa163 were called Core Sequence Promoters (COAs).Next, these four COAs were digested with PstI and XbaI and resultingfragments were ligated with a fragment of 4482 bp, which was excisedfrom pLUC-pro with PstI and XbaI. The promoter activity of these fourplasmids was measured as described in Example 1-1. In consequence, theactivity of pULC-COA273, pLUC-COA211, pULC-COA175 and pLUC-COA163 was97%, 76%, 96%, and 34%, respectively, of that of the full-lengthbeta-actin promoter. The promoter included in pULC-COA273 was designatedthe COA Promoter, since it showed the highest promoter activity.

[0040] 1-3 Construction of p45/46Sfi (FIGS. 2 & 3)

[0041] Based on the information of the gC homologue (gCh) gene of MDVserotype 1 (Coussens et al., J. Virol. 62:2373-2379, 1988) and itsadjacent BamHI-B fragment (Japanese Unexamined Patent Publication No.H6-292583), a DNA fragment having an SfiI site between two ORFs UL45hand UL46h, was prepared by PCR and cloned into pUC18. First, HVT DNA wasprepared from CEF cells infected with the HVT FC126 strain according tothe method of Lee et al. (J. Gen. Virol., 51: 245-253, 1980). Using theobtained HVT DNA as a template, PCR was performed with two pairs ofprimers. The first pair was SEQ NO. 7 (5′-CCCCGAATTCATGGAAGAAATTTCC-3′)and SEQ NO. 8 (5′-CGCGGGCCTTATTGGCCAAAACACACCTCTAACGGTTACT-3′). Thesecond pair was SEQ NO. 9(5′-GCGCGGCCAATAAGGCCAAAACACAGTAACCGTTAGAGGT-3′) and SEQ NO. 10(5′-CCCCAAGCTTTCAAGTGATACTGCGTGA-3′). Using the mixture of the obtainedtwo PCR products as template, another PCR was conducted with SEQ NO.7and SEQ NO. 8 to generate a fragment having an SfiI site between twoORFs UL45h and UL46h.

[0042] The resulting fragment was then digested with EcoRI and HindIIIand ligated to pUC18, which had been digested with EcoRI and HindIII.The obtained plasmid was designated p45/46Sfi.

[0043] 1-4 Construction of pUC18polyASfi

[0044] Plasmid pUC18polyASfi was constructed by annealing two syntheticoligonucleotides (SEQ NO. 11 and SEQ NO. 12) followed by ligation topUC18, which had been digested with EcoRI and HindIII. 138bp: SEQ NO. 115′-AGCTTGCCAATAAGGCTGCAGGTCGACTCTAGAGGATCCCCGGGCGAGCTCGCTAGCGGGCCCGCATGCGGTACCGTCGACAATAAAGAACCGCTTTAAGAATAGTGTTTATTTTTGTGTTTATGGCCAATAAGGCCG-3′ 138bp SEQ NO. 125′-AATTCGGCCTTATTGGCCATAAACACAAAAATAAACACTATTCTTAAAGCGGTTCTTTATTGTCGACGGTACCGCATGCGGGCCCGCTAGCGAGCTCGCCCGGGGATCCTCTAGAGTCGACCTGCAGCCTTATTGGC-3′

[0045] The inserted fragment was excised by BglI and the cohesive endswere designed to be inserted into the SfiI site of p45/46Sfi. Inaddition, a multi-cloning site was added to the 5′-terminus of thefragment so that a promoter-foreign gene cassette could be inserted. The43 bp sequence downstream from the SalI site was polyadenlyation signal,which was derived from the sequence located downstream of UL46h of theMDV GA strain.

[0046] 1-5 Construction of pBac

[0047] An DNA fragment of about 1.5 kilobases (kb) including thebeta-actin promoter described in EXAMPLE 1-1 was digested with PstI andSalI and ligated with pUC18polyASfi, which had been digested with PstIand SalI. The obtained plasmid was designated pBac.

[0048] 1-6 Construction of pGICOA

[0049] Using pBac as a template, PCR was performed with primers PrBac3and PrBac4. PrBac3: 5′-TTTCTGCAGTATTTTGTGCAGCGAT-3′ (SEQ NO. 13) PrBac4:5′-CTGTCTAGATAACGCGGTCAGTCAGA-3′ (SEQ NO. 14)

[0050] PrBac3 has a PstI site and PrBac4 has an XbaI site. ThePCR-amplified fragment was excised with PstI and XbaI to generate afragment of about 300 bp. The fragment was then ligated topUC18polyASfi, which had been digested with PstI and XbaI. The obtainedplasmid was designated pGICOA.

[0051] 1-7 Construction of Pec Promoter

[0052] The CMV enhancer was added to the COA promoter to enhance itspromoter activity.

[0053] Since the CMV enhancer had two BglI sites, a BglI cassette fromthe CMV enhancer was not easily inserted into the SfiI site of pGICOA.To delete BglI sites, in vitro mutagenesis was conducted by PCR withthree pairs of primers using pBK-CMV (Stratagene) as a template. Theprimers were PrCMV1 (SEQ NO. 15) and PrCMV3 (SEQ NO. 17), PrCMV4 (SEQNO. 18) and PrCMV5 (SEQ NO. 19), and PrCMV6 (SEQ NO. 20) and PrCMV2 (SEQNO. 16). Using the obtained three amplified fragments as a template, thesecondary PCR was performed with primers PrCMV1 and PrCMV2. Since PrCMV1had a PstI site and PrCMV2 had an EcoT22I site, the amplified fragmentwas digested with PstI and EcoT22I to yield a fragment of about 300 bp,which was in turn cloned into the PstI site of pGICOA to generatepGIPec. The promoter included in pGIPec, designated Pec promoter,consisted of about 275 bp fragment from the CMV enhancer followed by a273 bp fragment from the beta-actin promoter. The Pec promoter showedenhanced promoter activity, 6.5 times higher than that of COA promoterwhen evaluated in vitro as described in Example 1-1. PrCMV1: (SEQ No.15) 5′-GGG CTG CAG AGT TAT TAA TAG TAA TCA ATT-3′ PrCMV2: (SEQ No. 16)5′-GGG ATG CAT CCA TTT ACC GTC ATT GAC GTC-3′ PrCMV3: (SEQ No. 17)5′-GGG TCG TTG GGC GGT CAG CCG GCG G-3′ PrCMV4: (SEQ No. 18) 5′-CTT ACGGTA AAT GGC CCG CCC GCT G-3′ PrCMV5: (SEQ No. 19) 5′-TAC ACT TGA TGT ACTGCC AAT GGG C-3′ PrCMV6: (SEQ No. 20) 5′-TAT TTA CGG TAA ACT CCC CAT TGGC-3′

[0054] 1-8 Construction of NDV-F Vector

[0055] Using XLIII10H (Sato, H. et al., Virus Research, 7241-7255, 1987)as a template, PCR was performed with primers PrF1 and PrF2. PrF1:5′-GCTCTAGAGGATCCGCATGGGCTCCAGATCTTCTACCAGGATCCC-3′ (SEQ No. 21) PrF2:5′-GCGAGCTCGGTCCATGACTGAAGACTGCTATTGG-3′ (SEQ No. 22)

[0056] PrF1 has XbaI and BamHI sites. PrF2 has a SacI site.

[0057] The amplified fragment of about 1.9 kb long encoded 553 aminoacids of the F gene, which was identical to that reported in VirusResearch, 7241-7255, 1987. The fragment was digested with XbaI and KpnIand cloned into pGIPec, which had been digested with XbaI and KpnI. Theobtained plasmid was designated pGIPecF.

[0058] 1-9 Construction of the Homology Vector p45/46PecF

[0059] For construction of the homology plasmid, p45/46pecF, the Pecpromoter, the NDV F gene and the SV40 polyadenylation signal sequencewas inserted in p45/46Sfi. First, the Pec promoter and the NDV F genewere excised from pGIPecF with BglI and KpnI. Second, the SV40polyadenylation signal sequence was amplified from pBR-CMV (Stratagene)by PCR and cut with BglI and KpnI. These two fragments were cloned intothe SfiI site disrupting one of the SfiI sites and resulting in thehomology plasmid, p45/46pecF.

EXAMPLE 2 Construction and Purification of rHVT/NDV

[0060] Viral DNA of the HVT wild type, FC126 strain (wt-HVT) wasprepared as described by Morgan et at. (Avian Diseases, 34:345-351,1990). The CEF cells were transfected with the prepared wt-HVT DNA andp45/46PecF (see Example 3-3). The resulting recombinant virus was plaquepurified by staining plaques with the anti-NDV-F antibody.

[0061] Briefly, 5 μg of the homology vector p45/46PecF and 25 μg ofwt-HVT DNA were dissolved in 100 μl of Saline G (0.14 M NaCl, 0.5 mMKCl, 1.1 mM Na₂HPO₄, 1.5 mM NaH₂PO4, 0.5 mM MgCl₂, 0.011% glucose).Next, CEF cells were suspended in 0.7 ml of Saline G and subjected toelectroporation. Transfected cells were incubated for 10 minutes at roomtemperature and transferred to a 60-mm dish, which contained 5 ml mediumconsisting of Leibovitz's L-15 (GIBCO BRL, Cat. #41300-39) and McCoy's5A Medium (GIBCO BRL, Cat. #21500-061) (1:1) and 4% calf serum (LM (+)medium). After incubating at 37° C. in 5% CO₂, recombinants werepurified from wt-HVT by a series of limiting dilutions. Expression ofthe F gene was confirmed at each round of purification using anantigen-antibody reaction using the anti-NDV-F monoclonal antibody3-1G/5 (Morrison, T. G., Proc. Natl. Acad. Sci. U.S.A. 84: 1020-1024,1987) as the primary antibody. The purification procedure was repeateduntil every obtained plaque was stained positively by the anti-NDV-Fantibody. The purified recombinant HVT was designated rHVT/NDV.

EXAMPLE 3 Verification of the Stability of rHVT/NDV

[0062] 3-1 Southern Hybridization

[0063] The purified rHVT/NDV was propagated on CEF cells of two 150-mmdishes to obtain confluent plaques. Cells were recovered from dishes byscraping, transferred to Falcon tubes and centrifuged at 1,500revolutions per minute (rpm) for 5 minutes. Harvested cells were washedwith phosphate buffered saline (PBS), re-suspended in 1.2 ml of PBS and0.8 ml of a lysis buffer (1.25% TritonX-100, 250 mM 2-ME, and 50 mM EDTAin PBS) and lysed by vortexing for 30 seconds. The lysates were thencentrifuged at 3,000 rpm for 5 minutes at room temperature and thesupernatant was transferred to an Eppendorf tube. The viruses werecollected by centrifugation at 15,000 rpm, 22° C. for 20 minutes. Therecovered pellets were then suspended in 1 ml of 12.5 mM Tris-Cl (pH7.5) supplemented with 4 μl of a nuclease solution (0.25 mg/ml DNase 1,0.25 mg/ml RNase A, 150 mM NaCl), incubated at 37° C. for 30 minutes,and disrupted by incubating at 55° C. for 30 minutes with a SDS-proteasesolution (500 mM EDTA, 25 μl; 10% SDS, 125 μl; H₂O, 87 μl; 10 mg/mlProtease K, 12.5 μl; 2-Mercaptoethanol, 0.5 μl). The obtained mixturewas treated twice with phenol-chloroform, and 16 μl of 5M NaCl was addedto the aqueous phase. The viral DNA was precipitated by adding 2.5volumes of −20° C. ethanol, washed with 70% ethanol and centrifuged.After air-drying, the recovered pellets were dissolved in 50 μl of TEbuffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA).

[0064] The recovered viral DNA in TE buffer was then digested with XhoI,XbaI, and SfuI and subjected to 0.8% agarose gel electrophoresis. Theelectrophoresed DNA fragments on the single gel were transferredsimultaneously to two nylon membranes (Molecular Cloning: A LaboratoryManual, third edition, 6.35, Sambrook, J., and Russell, D. W. ColdSpring Harbor Laboratory). After fixing DNA by baking, the immobilizedDNA was hybridized with a DIG-labeled probe, “F probe” or “IS45/46probe”, which was prepared with PCR DIG Probe Synthesis Kit (ROCHEDIAGNOSTICS, Cat. #1636090). The F probe was prepared by PCR withNDV-F-F (SEQ ID NO. 23) and NDV-F-R (SEQ NO. 24) as primers andp45/46PecF as a template. IS45/46 probe was prepared with 45/46-F (SEQNO. 25) and 45/46-R (SEQ NO. 26) and p45/46Sfi. NDV-F-F5′-CTAGCAGTGGCAGTTGGGAAGAT-3′ ≡SEQ NO. 23≡ NDV-F-R5′-GTTAAGGCAGGGGAAGTGATTTGT-3′ ≡SEQ NO. 24≡ 45/46-F5′-GGGGAAGTCTTCCGGTTAAGGGAC-3′ ≡SEQ NO. 25≡ 45/46-R5′-GGTGCAATTCGTAAGACCGATGGG-3′ ≡SEQ NO. 26≡

[0065] The results of southern blotting showed that a 3.6 kb fragmenthybridized to the F probe and 3.6 and 1.2 kb fragments hybridized to theIS45/46 probe, indicating that the obtained rHVT/NDV had the expectedgenomic structure.

[0066] 3-2 In vitro Stability of rHVT/NDV

[0067] rHVT/NDV was passaged ten times in CEF cells and subjected toSouthern blot analysis. The results were the same with those obtained inExample 3-1, indicating that rHVT/NDV of the present invention wasstable even after the 10 passages.

[0068] 3-3 In vivo Stability of rHVT/NDV

[0069] 3000 PFU of rHVT/NDV was inoculated subcutaneously into the backof the SPF chicken or of commercial chicken having the anti-NDV maternalantibody. At week 3, 4, 5, and 6 post vaccination, peripheral blood wascollected from the vaccinated birds and viruses were recovered fromlymphocytes in the peripheral blood. Harvesting the lymphocytes wasperformed as follows.

[0070] Using a 2.5 ml syringe containing 0.5 ml of a heparin solution(100 u/ml), 2 ml of blood was collected from chickens and placed over 5ml of Ficoll-Paque (Amersham-Pharmacia) in a 15 ml Falcon centrifugetube. After centrifugation at 1800 rpm for 20 minutes, a band ofperipheral blood lymphocytes was formed at the boundary between theFicoll-Paque and serum layers. The boundary layer was collected with aPasteur pipette, inoculated into CEF cells in a 9 cm dish and cultivatedfor seven days. Isolation of viruses from lymphocytes was consideredsuccessful when plaques were observed during the process of the firstcultivation followed by the subcultivation. The results were summarizedin Table 1. TABLE 1 Isolation of rHVT from peripheral blood lymphocytesof vaccinated birds rHVT/NDV HVT FC126 Non-vaccinated MA+*** SPF**** MA+SPF MA+ 3 wks** 2/2* ND***** 2/2 1/1 ND 4 wks 3/3 2/2 1/3 2/2 0/2 5 wks3/3 2/2 1/3 ND 0/2 6 wks 3/3 2/2 1/3 2/2 0/2

[0071] As shown in Table 1, rHVT/NDV as well as the parent HVT FC126strain was recovered from peripheral blood lymphocytes of vaccinatedchickens from three to six weeks post inoculation. When stained with theanti-F antibody as described in Example 2, all recombinant plaques wereshown to express the F gene, indicating that rHVT/NDV was stable afterin vivo passage.

EXAMPLE 4 Verification of the Inserted Gene Expression by rHVT/NDV

[0072] 4-1 Immunofluorescence Technique

[0073] rHVT/NDV-infected cells were incubated with fresh CEF cells intissue culture chamber slides at 37° C. and obtained plaques were fixedwith cold acetone. To detect the expressed antigen gene products, thechicken anti-NDV antiserum or rabbit anti-F antiserum was used in the500-fold dilution in PBS as the primary antibody. Fixed cells wereincubated with the primary antibody at room temperature in 100% humidityfor about one hour, washed three times with PBS, and reacted with thesecondary antibody for about one hour at room temperature. Theanti-chicken immunoglobulin or anti-rabbit IgG antibody, which waslabeled with a fluorescent substance (FITC), was used as the secondaryantibody. After washing three times with PBS, the treated slides wereinspected by fluorescence microscopy. Cells infected with parent HVTFC-126 were used as a control. The results were summarized in Table 2.TABLE 2 Expression of the inserted F gene by rHVT/NDV (Detection offluorescence) Primary antibody anti-VP2 rabbit anti-F chicken anti-NDVmonoclonal Virus antiserum antiserum antibody ▪R63 ▪ PBS rHVT/NDV + + −− FC126 − − − − None − − − −

[0074] As shown in Table 2, rHVT/NDV expressed the inserted NDV-F gene.

[0075] 4-2 Western Blotting

[0076] CEF cells were infected with rHVT/NDV at m.o.i.=0.1, incubatedfor 72 hours, and solubilized in a SDS-GEL loading buffer. Similarly,cells infected with parent HVT FC126 or non-infected cells wereincubated and solubilized. The obtained samples were reduced, denatured,and subjected to SDS-PAGE. The electrophoresed proteins were transferredfrom SDS-GEL to a PVDF membrane (Immobilon-P, Millipore), which wasblocked in 1% w/v non-fat milk powder in PBS at room temperature for onehour. The treated membrane was then reacted with the anti-F rabbitantiserum in 500-fold dilution at room temperature, washed three timeswith PBS, and incubated for one hour with the biotinylated anti-rabbitgoat antiserum. After washing three times with PBS, the membrane wasincubated for one hour with an avidin-alkaline phosphatase complex,washed three times with PBS and one time with TBS, and reacted withBCIP-NBT (a substrate of alkaline phosphatase.) As shown in FIG. 4, aprotein band of 60 kilodaltons (kDa) was observed only in the lane withrHVT/NDV infected cells, which was the expected size of the F protein.

EXAMPLE 5 Efficacy of rHVT/NDV in SPF Chickens

[0077] rHVT/NDV obtained in Example 2 was subjected to the efficacy testas a Newcastle disease vaccine.

[0078] 1,950 PFU/100 μl/bird of rHVT/NDV were inoculated subcutaneouslyinto the back of fifteen one-day-old SPF chickens (LineM, JapanBiological Laboratories) using 20Gauge syringe. From three weeks postvaccination onward, the serum was collected from the vaccinated birdsand anti-NDV antibody titer was measured by a commercial ELISA kit(IDEXX, ELISA kit to diagnose Newcastle Disease). Chickens of thepositive control group were vaccinated at 14 day of age with acommercial NDV live vaccine according to the vender's recommendation.Chickens of the negative control group were not administered with anyvaccine. At 43 days of age (42 days post vaccination), chickens of allthree groups were challenged with 10³EID₅₀ of NDV-TexasGB, the standardchallenge strain in the United States, by intramuscular route to thefemoral region. The challenged chickens were inspected daily to checkmortality or to detect any onset of Newcastle disease. TABLE 3 Challengeexperiments of rHVT/NDV-vaccinated SPF chickens with virulent NDV DoseNo. of (PFU/ No. of symptom/total HI (ELISA) titer ELISA titer atVaccination chicken) chickens (%) at hatch challenge rHVT/NDV 1950 20 0/20 (0) 0 0.207 ± 0.03 Commercial NDV On label 10  0/10 (0) 1.089 ±0.29 Live vaccine Positive Controls N/A 10 11/12 (92) 0.089 ± 0.01Negative Controls N/A 5  0/5 (0) N/A

[0079] As shown in Table 3, chickens vaccinated with rHVT/NDV did notshow any clinical signs and the ELISA titer at the day of challenge wassignificantly elevated.

EXAMPLE 6 Efficacy of rHVT/NDV in NDV Maternal Antibody PositiveChickens

[0080] To examine the efficacy of rHVT/NDV as a vaccine with anti-NDVmaternal antibody positive chickens, fertilized eggs of commercialchickens (Hy-line, Kanagawa Youkei Rengoukai) were purchased andincubated. 1,950 PFU/μl of rHVT/NDV were inoculated into 18-day-oldembryos using a 20Gauge-1.5 inch syringe. Chickens of the positivecontrol group were vaccinated at 14 days of age with a commercial NDVlive vaccine according to the vender's recommendation. Chickens of thenegative control group were not administered with any vaccine. At 43days of age, chickens of all groups were challenged with 10³EID₅₀ ofNDV-TexasGB, the standard challenge strain in the United States, byintra-muscular route to the femoral region. The challenged birds wereinspected daily to check the mortality or to detect any onset ofNewcastle disease. TABLE 4 Challenge experiments of rHVT-vaccinatedcommercial chickens with virulent NDV Dose No. of (PFU/ No. ofsymptom/total HI (ELISA) titer ELISA titer at Vaccination chicken)chickens (%) at hatch challenge rHVT/NDV 1950 10  0/12 (0) N/A 0.471 ±0.108 Commercial NDV On label 10  0/10 (0) N/A 1.386 ± 0.287 Livevaccine Positive Controls N/A 10 10/10 (100) N/A 0.047 ± 0.003 NegativeControls N/A 5  0/5 (0) N/A N/A

EXAMPLE 7 Co-Relation Between the ELISA Titer and Vaccine Efficacy

[0081] Co-relation between the ELISA titer and protection was assessedby measuring the anti-NDV titer in the sera collected at the day ofchallenge from chickens, which were vaccinated as described in Example 5and 6. The antibody titer was measured by the commercial ELISA kitdescribed in Example 5. FIGS. 5 and 6 show the ELISA titers of survivedor non-survived SPF and commercial birds, respectively. As indicated,all chickens having 0.15 or more of the antibody titer (S/P value)survived the virulent NDV challenge.

EXAMPLE 8 Duration of Immunity

[0082] Five chickens vaccinated with rHVT/NDV as described in Example 6were kept without challenge and every two weeks the NDV-ELISA titers ofthe collected sera were measured. As a control, non-vaccinated chickenswere subjected to the same procedure. The S/P value 0.15, obtained inExample 7, was used as a criterion to determine the protection. Theresults are shown in Table 5. TABLE 5 Duration of protective immunityconferred by rHVT/NDV Weeks of age Group 2 3 4 5 7 11 15 20 24 30 45 50rHVT/NDV antibody titer 0.85 0.50 0.31 0.21 0.36 0.84 0.91 0.88 1.210.90 1.04 1.12 protection Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes YesYes Non-vaccinated antibody titer 0.74 0.46 0.10 0.03 −0.01   0.02 0.050.02 0.02 0.00 0.01 0.02 Controls protection Yes Yes No No No No No NoNo No No No

[0083] As shown in Table 5, at 4 weeks of age, the ELISA titer of thenon-vaccinated chickens decreased to the value lower than the criticalone, indicating no protection. By 50 weeks of age, the titer decreasedto nearly zero. On the contrary, the vaccinated chickens showed thelowest titer at 5 weeks of age, which was high enough to confer thecomplete protection. Afterwards, the value continued to increasegradually to 1.21 at 24 weeks of age, and remained the same until 50weeks of age. These data indicate that the rHVT/NDV of the presentinvention is capable of inducing long-lasting protective immunity in thevaccinated maternal antibody-positive commercial birds.

EXAMPLE 9 Isolation of rHVT/NDV from Peripheral Blood Lymphocytes ofrHVT/NDV Vaccinated Chickens

[0084] 3000 PFU of rHVT/NDV was inoculated subcutaneously into the backof a day old anti-NDV maternal antibody positive commercial chickens.The chickens were kept for 30 weeks and every two weeks, peripheralblood was collected from the vein of the wing web of the vaccinatedbirds. Viruses were recovered from lymphocytes in peripheral blood asdescribed in Example 3. Viruses were recovered from all chickensvaccinated with rHVT/NDV and all viruses were shown to express the Fgene.

1 26 1 555 DNA Gallus gallus promoter (1)..(555) 1 gagttattaa tagtaatcaattacggggtc attagttcat agcccatata tggagttccg 60 cgttacataa cttacggtaaattggcccgc cggctgaccg cccaacgacc cccgcccatt 120 gacgtcaata atgacgtatgttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtggag tatttacggtaaactgccca ttggcagtac atcaagtgta tcatatgcca 240 agtacgcccc ctattgacgtcaatgacggt aaatggatgc agtattttgt gcagcgatgg 300 gggcgggggg ggggggcgcgcgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 360 gaggcggaga ggtgcggcggcagccaatca gagcggcgcg ctccgaaagt ttccttttat 420 ggcgaggcgg cggcggcggcggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 480 tgcgcgctgc cttcgccccgtgccccgctc cgccgccgcc tcgcgccgcc cgccccggct 540 ctgactgacc gcgtc 555 239 DNA Artificial Sequence Description of Artificial Sequencesyntheticprimer DNA for PCR 2 cagtgtcgct gcagctcagt gcatgcacgc tcattgccc 39 3 50DNA Artificial Sequence Description of Artificial Sequencesyntheticprimer DNA for PCR 3 gctctagagt cgacaagctt catggctggc tgcggaggaacagagaaggg 50 4 16 DNA Artificial Sequence Description of ArtificialSequencesynthetic primer DNA for PCR 4 tattttgtgc agcgat 16 5 25 DNAArtificial Sequence Description of Artificial Sequencesynthetic primerDNA for PCR 5 acgtctagaa ggcaacgcag cgact 25 6 26 DNA ArtificialSequence Description of Artificial Sequencesynthetic primer DNA for PCR6 ctgtctagat aacgcggtca gtcaga 26 7 25 DNA Artificial SequenceDescription of Artificial Sequencesynthetic primer DNA for PCR 7ccccgaattc atggaagaaa tttcc 25 8 40 DNA Artificial Sequence Descriptionof Artificial Sequencesynthetic primer DNA for PCR 8 cgcgggccttattggccaaa acacacctct aacggttact 40 9 40 DNA Artificial SequenceDescription of Artificial Sequencesynthetic primer DNA for PCR 9gcgcggccaa taaggccaaa acacagtaac cgttagaggt 40 10 28 DNA ArtificialSequence Description of Artificial Sequencesynthetic primer DNA for PCR10 ccccaagctt tcaagtgata ctgcgtga 28 11 138 DNA Artificial SequenceDescription of Artificial SequenceModified synthetic oligonucleotidefrom pUC18 11 agcttgccaa taaggctgca ggtcgactct agaggatccc cgggcgagctcgctagcggg 60 cccgcatgcg gtaccgtcga caataaagaa ccgctttaag aatagtgtttatttttgtgt 120 ttatggccaa taaggccg 138 12 137 DNA Artificial SequenceDescription of Artificial SequenceModified synthetic oligonucleotidefrom pUC18 plasmid 12 aattcggcct tattggccat aaacacaaaa ataaacactattcttaaagc ggttctttat 60 tgtcgacggt accgcatgcg ggcccgctag cgagctcgcccggggatcct ctagagtcga 120 cctgcagcct tattggc 137 13 25 DNA ArtificialSequence Description of Artificial SequenceSynthetic primer DNA for PCR13 tttctgcagt attttgtgca gcgat 25 14 26 DNA Artificial SequenceDescription of Artificial SequenceSynthetic primer DNA for PCR 14ctgtctagat aacgcggtca gtcaga 26 15 30 DNA Artificial SequenceDescription of Artificial SequenceSynthetic primer DNA for PCR 15gggctgcaga gttattaata gtaatcaatt 30 16 30 DNA Artificial SequenceDescription of Artificial SequenceSynthetic primer DNA for PCR 16gggatgcatc catttaccgt cattgacgtc 30 17 25 DNA Artificial SequenceDescription of Artificial SequenceSynthetic primer DNA for PCR 17gggtcgttgg gcggtcagcc ggcgg 25 18 25 DNA Artificial Sequence Descriptionof Artificial SequenceSynthetic primer DNA for PCR 18 cttacggtaaatggcccgcc ggctg 25 19 24 DNA Artificial Sequence Description ofArtificial SequenceSynthetic primer DNA for PCR 19 tacacttgat gtactgccaagggc 24 20 25 DNA Artificial Sequence Description of ArtificialSequenceSynthetic primer DNA for PCR 20 tatttacggt aaactgccca ttggc 2521 45 DNA Artificial Sequence Description of ArtificialSequenceSynthetic primer DNA for PCR 21 gctctagagg atccgcatgg gctccagatcttctaccagg atccc 45 22 34 DNA Artificial Sequence Description ofArtificial SequenceSynthetic primer DNA for PCR 22 gcgagctcgg tccatgactgaagactgcta ttgg 34 23 23 DNA Artificial Sequence Description ofArtificial SequenceSynthetic primer DNA for PCR 23 ctagcagtgg cagttgggaagat 23 24 24 DNA Artificial Sequence Description of ArtificialSequenceSynthetic primer DNA for PCR 24 gttaaggcag gggaagtgat ttgt 24 2524 DNA Artificial Sequence Description of Artificial SequenceSyntheticprimer DNA for PCR 25 ggggaagtct tccggttaag ggac 24 26 24 DNA ArtificialSequence Description of Artificial SequenceSynthetic primer DNA for PCR26 ggtgcaattc gtaagaccga tggg 24

What are claimed are:
 1. A recombinant herpesvirus of turkeys harboringan F protein gene of Newcastle disease virus under the control of apromoter of which sequence is shown in SEQ NO.1.
 2. A recombinantherpesvirus of turkeys as in claim 1 wherein the promoter and F proteingene are inserted into the noncoding, inter-ORF region of the backbonevirus genome.
 3. A recombinant herpesvirus of turkeys as in claim 2wherein the said noncoding region is that located between UL45 and UL46of the herpesvirus genome.
 4. A method of inducing protective immunityin an avian host against avian herpesvirus and Newcastle disease virus,which method comprises inoculating the avian host with the recombinantherpesvirus of turkeys as in claim 1, 2 or
 3. 5. A method of inducingprotective immunity in an avian host as in claim 4 wherein a recombinantherpesvirus of turkeys is administered to the avian host by subcutanousor in ovo route.
 6. A poultry vaccine comprising a recombinantherpesvirus of turkeys as in claim 1, 2, or 3.